Benefits of PEGylation: certolizumab pegol. Francesco M. Veronese Department of Pharmaceutical Sciences Padova University

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1 Benefits of PEGylation: certolizumab pegol Francesco M. Veronese Department of Pharmaceutical Sciences Padova University

2 nanotechnology Biomedicines and nanomedicines represent a turning point in drug therapy

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5 Main limits to an extensive use of peptides Proteolytic degradation Rapid renal excretion Immunogenicity Antigenicity Aggregation Solubility Difficult formulation and proteins in therapy A SOLUTION: to increase the size and to change the protein surface by the covalent linking of hydrophylic polymers

6 Polymers may protect proteins, but what polymers? IgG site Protein Active site Binding site The end-functionalized olygomer m-peg (Davis, Abuchovski) overcomes the problems of the protein cross-linking

7 Poly(ethylene glycol), PEG CH 3 O CH 2 CH 2 O H n PEG, in its methoxy form, is the ideal polymer. It posses only one potentially reactive group. It is FDA approved for internal use. Furthermore: It presents water and organic solvent solubility Has great flexibility The strong binding of water molecules to polymer oxygen units increases the PEG size, yielding decreased kidney filtration and repulsion of other proteins Allows for a clean chemistry It is non toxic and non immunogenic It is present in drugs as excipient and in body cure products and foods

8 PEG-conjugates are nanomedicines According to the ESF,.nanomedicines are systems working from the molecular levels using engineering devices and nanostructures (from 1 to 100nm size) to chieve a medical benefit And as such PEG-conjugates must undergo specific Regulatory Guidelines regarding: characterization toxicity, reproducibility, etc, for marketing approval

9 Correlation plot between PEG molecular weight and half life in blood after intravenous administration. The data were obtained from the papers of Yamoka et al. and Nakaoka et al.

10 Pathways for PEG or PEG-conjugates escape from body Renal excretion = the rate is related to Mol Wt (remember the raptation mechanism) Bile excretion = prevalent for high Mol Wt Chain cleavage by oxidative processes = mainly related to P-450 or aldehyde dehydrogenase action Possible cleavage of one chain of branched PEG = by anchimeric assisted hydrolysis Proteolytic degradation of protein moiety = mainly carried out in liver

11 What about PEG and PEG-Protein Conjugates Safety? According to Bendele et al. (Toxicol. Sci. 1998), long term administration of conjugates at high doses in animals produces renal tubular vacuolation, but not associated with clinical alterations. According to Webster et al. (Drug. Metab. Disp. 2007), PEG itself at high doses or PEG-conjugates have a toxicological profile of very low concern and it is well tollerate at high doses following chronic and acute administration, it is excreted as such or following oxidation. Several medicines administered i.v.(busulfan, Etoposide,Lorazepan, Venoglobi-S, Aralast etc.) contain high doses of PEG as excipient.

12 Polymers may protect proteins, but what polymers? IgG site Protein Active site Binding site The end-functionalized olygomer m-peg (Davis, Abuchovski) overcomes the problems of the protein cross-linking

13 Which amino acids of a protein can be exploited for polymer binding? Serine Treonine OH Asparagine Glutamine CONH2

14 The conjugate is composed of: PEG------Linker------Protein and each must be properly characterized

15 Example of PEG chemical linkage to the protein NH2 mpeg O 2a N N Cl N Cl O mpeg-o-ch 2 -CH 2 C 2b H + Protein-NH 2 + 1) Protein-NH 2 2) NaCNBH 3 NH-Protein N mpeg O N mpeg -O-CH 2 -CH 2 -CH 2 -NH-Protein N Cl But herogeneous protein conjugates are obtained

16 Common limitations : multiple isomers α-interferon, random amino PEGylation :polydispersity of PEG

17 How can we get a site specific PEGylation? >By changing the reaction conditions (ph, temperature, solvent,etc.) >By changing the structure of PEG >By using enzymes in the conjugation

18 PEG conjugates on the market PEG CONJUGATES Type of PEGylation Year to market Disease With Proteins PEG-asparaginase (Oncaspar ) PEG-adenosine deaminase (Adagen ) Random, linear PEG 5 kda Random, linear PEG 5 kda 1994 Acute lymphoblastic Leukemia 1990 Severe combined immunodeficiency disease (SCID) PEG-interferon a2a (Pegasys ) Random, Branched PEG 40 kda 2002 Hepatitis C PEG-interferon a2b (PEG-Intron ) Random, linear PEG 12 kda 2000 Hepatitis C and clinical trials for cancer, multiple sclerosis, HIV/AIDS. PEG-G-CSF (pegfilgrastim, Neulasta ) PEG-growth hormone receptor antagonist (Pegvisomant, Somavert ) PEG-Anti-TNF-Fab (Certolizumab, Cimzia) With Oligonucleotides Branched PEG-anti-VEGF aptamer (Pegaptanib, Macugen ) Selective, linear PEG 20 kda Random, linear PEG 5 kda (genetic modified protein) PEG 20kDa Selective, branched PEG 40 kda 2002 Treating of neutropenia during chemotherapy Acromegaly Rheumatoid arthritis -Crohn s disease 2004 Macular degeneration (age-related)

19 Comparison of Pegasys and PEG-Intron pharmacokinetics Concentration (pg/ml) 1500 Mean Serum Concentration-Time-Profile following Multiple Dose s.c. Injections PEGASYS 1000 Algranati et al., AASLD 99, Poster 120 (PEGASYS) Glue et al, AASLD 1999 (PEG-Intron) 500 PEG-Intron Time (hours) week

20 Biochemical and chemical properties of the two PEGylated interferons PK parameter IFNα PEGASYS PEG- INTRON t 1/2 (h) Clearance 6000 ml/h ml/h 725 ml/h Absorption half-life (h)

21 But othe properties are improved as well in PEG-conjugated proteins as: solubility, stability, loss of immunogenicity, loss of aggregation propensity etc. and body distribution

22 Tumour targeting by EPR effect (Maeda 2000) The same tissue accumulation was observed to take place in different inflammed tissues (Garrod 2006, Wang 2004 etc.) and to be probably at the basis of improved targeting in various diseases as rheumatic

23 What what is the case of antibodies as drugs?

24 Pharmacokinetics of IgG, Fab, di-fab and PEG-Fab IgG FAB-PEG FAB difab

25 The full antibodies size is sufficient for EPR effect, this is the case of the anti-tnfa Infliximab:.Chimeric mouse-human mab Adalimumab:.Human IgG mab while Certulizumab pegol:.humanized Fab fragment-peg reaches the needed size thanks to the contribution of the bound polymer

26 Vision of different anti-tnfa agents From crystallography From chemistry

27 Comparative characteristics and pharmacology of anti-tnfa agents Characteristics and parameters Agent Infliximab Adalumimab Certolizumab pegol Structure Chimeric mouse-human IgG1 mab Recombinant human IgG1 mab PEGylated humanized anti-fab fragment of an anti-tnfa mab No Fc region Conjugate None None PEG2*: 2 20 kda chains Route of administration Intravenous Subcutaneous Subcutaneous Dose 3 5 mg/kg at weeks 0, 2 and 6, then 8-weekly Can be increased to 10 mg/kg or frequency increased 40 mg every second week (or weekly if necessary) (RA). An induction of up to 160 mg followed by 80 mg 2 weeks later is required for CD. An 80-mg initial dose followed by 40 mg 1 week later is required for psoriasis 400 mg every 2 weeks for first 4 weeks, then every 4 weeks Half-life (days) Major biomarkers affected CRP, inflammatory cytokines, anti-ccp, RF, MMPs, bone and cartilage markers, regulatory T- cells CRP, inflammatory cytokines, anti-ccp, RF, MMPs CRP CCP, cyclic citrullinated peptide; CD, Crohn s disease; CRP, C-reactive protein; Ig, immunoglobulin; mab, monoclonal antibodies; MMPs, matrix metalloproteinases; PEG, polyethylene glycol; RA, rheumatoid arthritis; RF, rheumatoid factor; TNF, tumour necrosis factor. *PEG2 is linked to the Fab cysteine thiol group.

28 Certulizumab pegol: Properties-1 >Fc region absent in the product >Fab fragment with a reactive SH, for specific PEG conjugation in the hinge region, is obtained by recombinant gene technology >Fab fragment is conveniently produced by E Coli >The short ipervariable complementary determinant region of Fab derives from a murine mab >The position of conjugation allows for the binding and neutralization of TNF >It binds both soluble and membrane bound TNF >A branched PEG 40k, constituted by two chains of 20k bound together, is linked to a single point of Fab

29 Different architecture of PEG linear PEG X branched PEG 2 X

30 The PEG structure (linear or branched) plays also a role in the biological behaviour of conjugates

31 PEGs reactive towards a thiol group Structure Thioreactive PEGs Properties PEG S S N PEG-pyridildisulphide The most specific towards thiol but yields a cleavable linkage by a reducing agent also in vivo. PEG N O PEG-maleimide PEG O S O CH CH 2 PEG-vinylsulfone Give stable linkage by double bond addition but may also react with amines at ph > 8. O PEG NH O I PEG-iodo acetamide Less reactive, not much used.

32 Approximate molecular weights (kda) of anti-tnfa/tnfa immune complexes at differing molar ratios (ligh scattering values, kd) Parameter Agent Infliximab Adalumimab Certolizumab pegol TNFa alone Antibody alone Antibody:TNFa ratio 1: Antibody:TNFa ratio 4:

33 Certulizumab pegol: Properties-2 >Certulizumab does not cross-link to TNFa trimer, as adulimumab and infliximab, to yield complexes with proinflammatory effect as PMNs degranulation and superoxide formation

34 Certulizumab pegol: Properties-3 A strong binding takes place between certulizumab pegol and TNFa Affinities of infliximab, adalimumab, and certolizumab pegol for soluble TNFa Parameter Agent Infliximab Adalumimab Certolizumab pegol Association rate constant, k a (M -1 s - 1 ) Dissociation rate constant, k d (s -1 ) Equilibrium constant, K D (k d /k a, pm)

35 Certulizumab pegol: Properties-4 >The absence of Fc region prevents complement fixation and cell lysis, as well as elimination by FcRn mediated transport >The non reactivity with FcRn seems to reduce also transport to milk and crossing of placenta >On the contrary of infliximab and adalimumab, certulizumab pegol does not induce antibody-dependent cell mediated cytotoxicity, complement-dependent cytotoxicity or apoptosis in cells bearing membrane-bound TNFa

36 Certulizubab pegol: Properties-5 >The high hydrodinamic volume of PEG allows for a long retention time in blood and inflammed tissues >It is distributed to the major organs, but not brain >There is no evident accumulation over time and repeated administrations >NMR studies demonstrated the elimination, at 84 days, of over 73% in urine and 18% in faeces >SDS studies demonstrated that in urine PEG of 40k is present. The polymer is no further metabolized, while FAB is released

37 Pharmacokinetic parameters of certolizumab pegol in humans and primates (cynomolgus monkeys) after intravenous or subcutaneous administration Parameter Humans Primates Central volume of distribution, V 1 (ml/kg) 45 (44 47) 46 (44 48) Total body clearance, CL (ml/h/kg) 0.17 ( ) 0.29 ( ) Peripheral volume of distribution, V 2 (ml/kg) 29 (25 33) 31 (26 37) Intercompartmental clearance, CLd (ml/h/kg) 0.69 ( ) 1.1 ( ) Elimination half-life, t ½ (h) 313 ( ) 192 ( ) Bioavailability, F 1 ( ) 1.1 ( ) Absorption rate constant, Ka (h 1 ) ( ) 0.11 ( )

38 Summarizing, preclinical studies showed : Complex Affinity Cyt.Secr. CDC Apoptos. Neutroph Format. TNF Inhibit(3) ADCC (1) death (2) ertulizumab dalimumad nfliximab tanercept / DC, Complement dependent cytotoxicity; ADCC, Antibody dependent cytotoxicity 1)of activated lynphosites and monocytes, (2) degranulation (3) following anti-tnf ncubation and lipopolysacc. stimulation

39 Certulizumab pegol: Properties-6 Several phase II and III clinical trials (PRECISE 1 and 2, DMARG, RAPID 1 and 2) demonstrated that: >Certulizumag pegol is convenient for both monotherapy or in combination with MTX >It presents an acceptable safety profile >Significantly reduces the signs of RA >It inhibits the progression of structural damages >The onset of benefit certulizumab pegol is rapid >The majority of adverse events are mild to moderate >The discontinuation due to adverse event is low, below 6% >Low incidence of pain or reaction at injection site >Minimal incidence of infections, similar to other anti- TNFa agents

40 But we must also underline the cost of these nanomedicines, a problem that however is becoming common not only for the new proteins as drugs but for all of the drugs in general, due to: >>The research investments >>The increasing requirements by FDA or EMEA WE can say that now aspirin would not be approved by Regulatory Authorities

41 Thanks To conclude PEG is a wonderful and versatile tool for protecting and improving the properties of different drugs, as in nature spines are protecting the hedge horn, and we may be confident that this technique will give further Protein protection by PEGylation resembles the important applications in the near future hedge hog defense by spines, but how to PEGylate?

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